The long-range objective of the proposed research is to understand the molecular mechanisms that mediate the translocation of nascent secretory proteins across the membrane of the endoplasmic reticulum (ER), as well as the integration of nascent membrane proteins. Very little is now known about the environment and interactions of the nascent chains as they pass through, or are incorporated into, the membrane at sites termed translocons. The proposed experiments will examine these processes from a unique perspective, that of the nascent chain, by taking advantage of a class of Lys-tRNA analogs that were devised and developed by us. Fluorescent dyes or photoreactive moieties, covalently attached to a lysine side chain, will be incorporated into nascent chains in an in vitro translation system; nascent chains of defined lengths will be prepared by using secretory or membrane protein mRNAs truncated at specific locations. This novel approach creates translocation or integration intermediates that have a fluorescent or photoreactive probe located at a specific site in a functional complex (since the nascent chain complex can be generated in situ only by active ribosomes and translocons). A nascent chain fluorophore will be located in the middle of the bilayer to determine whether the nascent chain crosses the membrane through an aqueous pore or through a nonpolar environment by examining the emission properties of the dye and its sensitivity to iodide ions. Singlet-singlet energy transfer will be used to determine the distance of closest approach between the nascent chain and phospholipids (hence the size of the putative pore), whether the ribosome leaves the membrane during the integration of multispanning membrane proteins, and the topology of signal recognition particle (SRP) complexes with the ribosome and nascent chain, and with the SRP receptor and ER membrane. Photocrosslinking between a probe in the transmembrane sequence of a nascent chain and an ER membrane protein will be monitored to determine when a transmembrane segment disengages from the translocon, and whether this is affected by the length of nascent chain, the orientation of the sequence in the membrane, the presence of the ribosome, or the presence of additional transmembrane sequences in multi-spanning proteins. The sequence dependence of signal sequence affinity for SRP will be determined spectroscopically using fluorescent-labeled SRP or nascent chains to monitor complex formation. The location and orientation of the signal sequence will be determined using a fluorescent or photoreactive probe incorporated into the signal sequence, and its interactions/fate during translocation and integration will be monitored by using fluorescence energy transfer, collisional quenching of emission, and photocrosslinking to ER membrane targets.